One-Step Multiplex PCR Assay for Detecting Streptococcus pneumoniae Serogroups/Types Covered by 13-Valent Pneumococcal Conjugate Vaccine (PCV13)

Abstract

The life-threatening illnesses caused by Streptococcus pneumoniae have been declined significantly after the use of pneumococcal conjugate vaccines. Continuous monitoring of the vaccine serogroups/types is necessary to follow the changing epidemiology of invasive pneumococcal diseases. Recently, the sequential multiplex PCR approach, which uses several different sets of reactions, has been commonly adopted for determining capsular serogroups/types of S. pneumoniae isolates. In our study, we focused on development of a one-step multiplex PCR assay detecting all 1, 3, 4, 5, 6A/B, 7F, 9V, 14, 18C, 19A, 19F and 23F serogroups/types targeted by PCV13. The content of multiplex PCR mix and the cycling conditions were optimized in a manner that allowed rapid and accurate serotyping of a pneumococcal isolate by performing only a single amplification reaction. In our study of 182 clinical isolates, the one-step multiplex PCR assay exhibited 100% sensitivity and specificity, suggesting that its utilization can significantly reduce the use of traditional antiserum method requiring expensive reagents.

Etiology of Pediatric Bacterial Meningitis Pre- and Post-PCV13 Introduction Among Children Under 5 Years Old in Lomé, Togo

“…For pneumococcal serotyping, CSF of LytA positive samples was added to TE buffer containing 0.08 g/mL of lysozyme (Sigma-L-6876) and 150 U/mL of mutanolysin (Sigma M-9901) and incubated for 1 hour at 37° C. Nucleic acid extraction was carried out following Qiagen DNA Mini-kit manufacturer instructions and purified DNA underwent sequential triplex real-time PCR assay to detect 21 capsular serotypes as described elsewhere [28]. Additionally, samples with CT values ≤32 were classified as nontypeable and underwent conventional multiplex PCR analysis [29]. Meningococcal gene targets included sacB, synD, synE, synG, xcbB, synF for serogroups A, B, C, W, X, and Y, respectively.…”

<p>Serotype Distribution and Antibiotic Susceptibility of <em>Streptococcus pneumoniae</em> Isolates in Tehran, Iran: A Surveillance Study</p>

“…The PCV13 serotype distribution was determined by the multiplex PCR assay, based on coskun-Ari study. 18 The primers were selected based on CDC recommendation. 19 Each reaction comprised four primer pairs, targeting serotype-specific genes, and an internal control, the cpsA gene as a conserved region located at the cps loci.…”

Serotyping of pneumococci: evaluation of the genetic approach and performance with clinical samples

“…In 2011, in Belgium, Jourdain et al (2011) used a 30-valent multiplex PCR composed of 7 consecutive PCRs and an agar-based detection system to determine the serotypes from nasopharyngeal pneumococci. In 2012, Coskun-Ari et al (2012) presented data on a 13-valent multiplex PCR using a 3% agarose gel for detection and determination of serotypes from random clinical samples from Turkey. In 2013, Raymond et al (2013) presented data on a PCR-based automated microarray assay evaluated with 166 cultured pneumococci, and in May 2013, the CDC updated its protocols regarding genetic serotyping (CDC, 2013).…”

“…We chose a multiplex PCR approach with 3 different primer sets and opted against the single multiplex PCR approach used by Coskun-Ari et al (2012) because discrimination between amplicons with less than 20-bp difference in length is error prone. Additionally, we decided against a probe based multiplex approach due to total cost.…”